The invention relates to methods for producing and purifying recombinant adeno-associated virus (rAAV). More particularly, it relates to methods for producing commercial grade rAAV at large scale where rAAV was generated in the absence of infectious helper virus. The methods employ a plurality of column purification steps that yield purified rAAV. One embodiment of the invention is a two-column purification system comprising purification over an anion exchange column and over an affinity column. In another embodiment, a cation exchange column purification step is included.
Gene delivery is a promising method for the treatment of acquired and inherited diseases. A number of viral-based systems for gene transfer purposes have been described, including adeno-associated virus (AAV)-based systems. AAV is a helper-dependent DNA parvovirus that belongs to the genus Dependovirus. AAV requires co-infection with an unrelated helper virus, e.g., adenovirus, herpes virus, or vaccinia, in order for a productive infection to occur. In the absence of a helper virus, AAV establishes a latent state by inserting its genome into a host cell chromosome. Subsequent infection by a helper virus rescues the integrated viral genome, which can then replicate to produce infectious viral progeny.
AAV has a wide host range and is able to replicate in cells from any species in the presence of a suitable helper virus. For example, human AAV will replicate in canine cells co-infected with a canine adenovirus. AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. For a review of AAV, see, e.g., Berns and Bohenzky (1987) Advances in Virus Research (Academic Press, Inc.) 32:243-307.
The AAV genome is composed of a linear, sing-stranded DNA molecule that contains 4681 bases (Berns and Bohenzky, supra). The genome includes inverted terminal repeats (ITRs) at each end that function in cis as origins of DNA replication and as packaging signals for the virus. The ITRs are approximately 145 bp in length. The internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV rep and cap regions, respectively. These regions code for the viral proteins that provide AAV helper functions, i.e., the proteins involved in replication and packaging of the virion. Specifically, a family of at least four viral proteins is synthesized from the AAV rep region, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2 and VP3. For a detailed description of the AAV genome, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129.
The construction of infectious recombinant AAV (rAAV) virions has been described. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Numbers WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.
Contemporary rAAV virion production involves introduction of an AAV vector plasmid and an AAV helper vector plasmid into a host cell. After the AAV helper plasmid and the AAV vector plasmid bearing the heterologous nucleotide sequence of interest are introduced into the host cell (generally by stable or transient transfection), the cells can be infected with a suitable helper virus in order to provide the required accessory functions. Most typically, the helper virus will be infectious adenovirus (type 2 or type 5) or herpes virus, and will, among other functions, transactivate the AAV promoters present on the helper plasmid directing the transcription and translation of AAV rep and cap regions.
AAV vectors can be engineered to carry a heterologous nucleotide sequence of interest (e.g., a selected gene, antisense nucleic acid molecule, ribozyme, or the like) by deleting, in whole or in part, the internal portion of the AAV genome and inserting the DNA sequence of interest between the ITRs. The ITRs remain functional in such vectors allowing replication and packaging of the rAAV containing the heterologous nucleotide sequence of interest. The heterologous nucleotide sequence is also typically linked to a promoter sequence capable of driving gene expression in the patient""s target cells under the certain conditions. Termination signals, such as polyadenylation sites, can also be included in the vector.
AAV helper functions can be provided in trans via an AAV helper vector. For example, such helper vectors can be plasmids that include the AAV rep and/or cap coding regions but which lack the AAV ITRs. Accordingly, such a helper vector could neither replicate nor package itself. A number of vectors that contain the rep coding region are known, including those vectors described in U.S. Pat. No. 5,139,941, having ATCC accession numbers 53222, 53223, 53224, 53225 and 53226. Similarly, methods of obtaining vectors containing the HHV-6 homologue of AAV rep are described in Thomson et al. (1994) Virology 204:304-311. A number of vectors containing the cap coding region have also been described, including those vectors described in U.S. Pat. No. 5,139,941.
After culturing the host cells with the necessary components for rAAV production, the host cell is harvested and a crude extract is produced. The resulting preparation will contain, among other components, approximately equal numbers of rAAV virion particles and infectious helper virions. Although rAAV virion particles produced via adenovirus infection can be purified away from some of the contaminating helper virus, unassembled viral proteins (from the helper virus and AAV capsid), and host cell proteins using known techniques, such preparations contain high levels of contaminants. Approximately 95% of the contaminants are derived from adenovirus, and 50% or greater of the total protein obtained in such rAAV virion preparations is free adenovirus fiber protein. Because free adenovirus fiber protein tends to co-purify with rAAV virions, this association makes separation of the two especially difficult, lowering rAAV virion purification efficiency. Moreover, adenovirus contaminants may be particularly problematic since many adenoviral proteins, including the fiber protein, have been shown to be cytotoxic and highly immunogenic. Preparations of rAAV containing adenoviral contaminations therefore may damage the target cell or provoke undesired immune responses in the host. Varying amounts of several other unidentified adenoviral and host cell proteins are also present in the preparations. Importantly, significant levels of infectious adenovirus virions are also obtained. To inactivate the infectious adenovirus, the preparation is heat inactivated (56xc2x0 C. for 1 hour). Heat inactivation, however, results in an approximately 50% drop in the titer of functional rAAV virions.
Therefore, production of rAAV virions using infectious helper viruses (such as adenovirus type-2, or a herpes virus) is undesirable for several reasons. Such production methods require the use and manipulation of large amounts of high titer infectious helper virus that present a number of health and safety concerns. Also, concomitant production of helper virus particles in rAAV producing cells diverts large amounts of cellular resources away from rAAV virion production likely lowering rAAV virion yields. Finally, the rAAV yields are even furthered lowered by the extensive purification required to remove contaminating infectious helper virus.
Current Purification Methods for rAAV
Several methods for purifying rAAV have been described in the literature. While rAAV purified by CsCl density gradients has been successfully used in human clinical trials, the method is not suitable for producing commercial scale quantities of rAAV. A method of purifying rAAV using only cationic column-chromatography has been described. However, the method fails to remove enough contaminating DNA and protein to be suitable for commercial use. A second chromatographic technique designed to purify rAAV produced using infectious adenovirus has also been described.
In order to effectively remove and/or inactivate potentially dangerous helper virus, this method involves several steps, including chromatography over anion or cation exchange columns as well as purification through tangential flow filtration or affinity purification. Even after extensive column purification, the resulting product typically will require heat inactivation to destroy any remaining helper virus particles at the expense of a substantial loss of rAAV, as much as 50%.
Because this purification system is designed primarily to remove infectious helper virus (particularly adenovirus), the system is not optimized to purify rAAV produced without infectious helper virus. Specifically, many steps are required and each of the steps results in loss of product rAAV virions, thus reducing the final yield of the recombinant product. Therefore, significant rAAV loss is unavoidable using such rAAV purification protocols increasing both the difficulty and cost of the procedure.
In sum, all prior purification systems have been designed primarily to remove and/or inactivate contaminating infectious helper virus, or are otherwise not suitable for commercial use. Thus, such systems are unsatisfactory for purifying rAAV produced in the absence of infectious helper virus, especially for large-scale production of commercial-grade rAAV. Consequently, there remains a need to provide a scalable and efficient purification system capable of separating contaminating protein and nucleic acid from rAAV prepared in the absence of infectious helper virus.
The present invention involves large-scale purification of recombinant AAV (rAAV) virions that were produced in the absence of infectious adenovirus. The methods include preparing a lysate from the host cell line then passing the lysate over various combinations of ion exchange chromatography media and/or affinity chromatography media. The host cell line is preferably cultured in roller bottles, a bioreactor, or using another technique suitable for large-scale cell culture.
In certain preferred embodiments, the rAAV is generated in a host cell line by triple-transfection with an accessory function vector, an AAV vector, and an AAV helper vector. The accessory function vector generally includes accessory functions provided by one or more adenovirus early region sequences, particularly, the E1b, E2a, E4, and/or VA RNA regions. The AAV vector includes one or more heterologous nucleotide sequences of interest flanked by at least one functional inverted terminal repeat (ITR) sequence. Finally, the AAV helper vector generally includes the AAV rep and/or the AAV cap sequences. In certain embodiments, the AAV helper vector may also have a modified AAV p5 promoter. After harvesting the transfected cell line, a lysate is formed using techniques suitable for large-scale production, such as microfluidization.
The affinity chromatography medium used for purifying rAAV will preferably be an AAV receptor or an antibody with binding affinity for AAV. In a preferred embodiment, heparin sulfate is used as the affinity chromatography medium. A variety of cation exchange and anion exchange media are contemplated for use with the present invention. Suitable cation exchange media include sulfo-, phospho, carboxy-, and carboxy-methyl-based resins. In one embodiment, a cation exchange chromatography module is used that contains a medium known as MUSTANG S(trademark). Suitable anion exchange media include N-charged amino or imino resins such as STREAMLINE Q XL(trademark), MUSTANG Q(trademark), POROS 50 PI, SEPHAROSE Q, any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins.
Several optional purification steps may also be used with the present methods, such as filtering the lysate through one or more filters or treating the lysate with a nuclease. In the certain embodiments, the methods are optimized to purify rAAV that was produced in a host cell line cultured in the absence of serum.